Process for the preparation of amides

ABSTRACT

A novel biotechnological process for the preparation of nitriles, starting from amides, is described. Micro-organisms of the genus  Amycolatopsis, Actinomadura  or  Rhodococcus  are employed for this process.

This application is a divisional of 09/463,203, filed Mar. 27, 2000, nowU.S. Pat. No. 6,444,451, which is a national phase application under 35U.S.C. §371 of International Application No. PCT/EP98/04587,incorporated by reference herein, which was filed on Jul. 22, 1998 andpublished in German on Feb. 4, 1999.

The invention relates to novel microorganisms of the genus Actinomadura,Amycolatopsis or Rhodococcus, and to a novel process for the preparationof amides using these microorganisms or using enzyme extracts of thesemicroorganisms.

For amides such as, for example, nicotinamide, a vitamin of the vitaminB complex which is essential to animals and man, a number ofbiotechnological processes are already known. Generally, it is knownthat microorganisms containing nitrile hydratase convert nitriles to thecorresponding amides. Thus EP-A-0 188 316 describes a process for thepreparation of nicotinamide starting from 3-cyanopyridine usingmicroorganisms of the genus Rhodococcus, Arthrobacter or Microbacterium.

A disadvantage of this process is that these micro-organisms have only alow activity for the conversion of 3-cyanopyridine to nicotinamide.

EP-A-0 307 926 describes the conversion of 3-cyano-pyridine tonicotinamide by means of microorganisms of the species Rhodococcusrhodochrous J1. In order that these microorganisms catalyse the desiredconversion, they must be induced.

A further disadvantage of this process is that Rhodococcus rhodochrousJ1 is red-coloured and accordingly a discoloration of the product takesplace. In addition, this microorganism has a low heat stability and isinhibited, for example, by the substrate 3-cyano-pyridine.

A further process for the preparation of nicotinamide starting from3-cyanopyridine by means of microorganisms of the species Rhodococcusrhodochrous J1 is described in EP-A-0 362 829. In order to increase thespecific activity of the microorganisms containing nitrile hydratase,urea or a urea derivative was added to the culturing medium as aninducer. As in the process described beforehand, a discoloration of theproduct also takes place in this process.

In addition, WO 95/17 505 describes a process for the preparation ofaromatic amides starting from the corresponding nitriles by means ofmicroorganisms of the species Rhodococcus rhodochrous M33. Adisadvantage of this process is the red colouration of Rhodococcusrhodochrous M33 and also the high K_(M) value for the substrate3-cyanopyridine.

The object of the present invention was to eliminate these disadvantagesand to make available a process for the preparation of amides in whichthe corresponding amides can be isolated in good yield and purity.

This object is achieved by the novel microorganisms according to claims1 and 3, and by the process according to claim 6.

According to the invention, the process is carried out by converting anitrile, as substrate, to the corresponding amide by means ofmicroorganisms of the genus Actinomadura, Amycolatopsis or Rhodococcus,using an enzyme extract of these microorganisms or by means of purifiednitrile hydratase of microorganisms of the genus Amycolatopsis orActinomadura.

The nitriles employed for the biotransformation such as, for example,3-cyanopyridine are commercially available compounds.

The microorganisms according to the invention are able to convertnitriles as substrates into the corresponding amides. Preferably, thesemicroorganisms have the ability to grow on nitriles or amides as thesole C and/or N source.

The microorganisms according to the invention are obtainable by means ofsuitable selection, for example, from soil samples, sludge or wastewater with the aid of customary microbiological techniques. Expediently,the microorganisms are selected by growth with nitriles or amides as thepreferably sole C and N source in the presence of cobalt ions. Nitrilesand amides suitable for selection are, in particular, the nitriles alsoemployed as substrates in the later biotransformation and thecorresponding amides obtainable therefrom. Suitable growth media arelikewise known to the person skilled in the art, for example the mediumdescribed in Table 1 can be used.

Customarily, the microorganisms are cultured (grown) in the same mannereven before the actual biotransformation, the abovementioned media beingused.

As known professionally, a nitrile hydratase is only formed when thegrowth medium contains cobalt ions as a cofactor. Suitable “cobaltcompounds generating cobalt ions” are Co²⁺ or Co³⁺ salts. Examples ofCo²⁺ and Co³⁺ salts are cobalt chlorides, cobalt sulphates and cobaltacetates.

Expediently, the cobalt compound employed is a Co²⁺ salt such as, forexample, CoCl₂. Growth, however, can also be carried out in the presenceof vitamin B12 together with metallic cobalt or other cobalt compoundswhich generate a cobalt ion in situ. Expediently, the cobalt compound isemployed in an amount from 1 to 10 mg/l, preferably from 1 to 3 mg/l.

Customarily, growth is carried out at a temperature from 20 to 50° C.and at a pH between pH 5 and pH 8, preferably from 30 to 45° C. andbetween pH 5.5 and pH 7.5.

The actual biotransformation can be carried out using microorganisms ofthe genus Actinomadura, Amycolatopsis, using an enzyme extract of thesemicroorganisms or by means of purified nitrile hydratase from thesemicroorganisms. Expediently, the biotransformation is carried out usingmicroorganisms of the species Actinomadura spadix, for example theisolates Actinomadura spadix E3733, Actinomadura spadix E3736,Actinomadura spadix 45A32, Actinomadura spadix 4501 or Actinomaduraspadix C15. The biotransformation is preferably carried out usingmicroorganisms corresponding to the species Amycolatopsis NE 31 andAmycolatopsis NA40 or their functionally equivalent variants andmutants. Micro-organisms corresponding to the species Amycolatopsis NA40are particularly preferably employed. Microorganisms of the speciesmentioned were deposited on 03.06.1997 in the Deutschen Sammlung vonMikroorganismen und Zellkulturen GmbH [German Collection ofMicroorganisms and Cell Cultures GmbH], Mascheroderweg 1b, D-38124Brunswick under the designations Amycolatopsis NE 31 and AmycolatopsisNA40 according to the Budapest Convention and have the deposit numbersDSMZ 11616 and DSMZ 11617 respectively. These two microorganisms havebeen more accurately identified and are to be assigned to species of thegenus Amycolatopsis which have not yet been described in the literature.

Accordingly, the invention also relates to microorganisms of the genusAmycolatopsis or Actinomadura which are capable of converting an amideinto a nitrile, in particular microorganisms with the designationAmycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616).

In addition, it has been found that specific microorganisms of the genusRhodoccus have better properties for the conversion of nitriles toamides than the Rhodococcus rhodochrous J1 described in EP-A-0 362 829.These microorganisms are Rhodoccus GF674, Rhodoccus GF578, RhodoccusGF473, Rhodoccus GF270 (DSMZ 12211) and Rhodoccus GF376 (DSMZ 12175) ortheir functionally equivalent variants and mutants. The microorganismDSMZ 12175 was deposited on 15.5.1998 and the microorganism DSMZ 12211on 8.6.1998 in the Deutschen Sannlung von Mikroorganismen undZellkulturen GmbH [German Collection of Microorganisms and Cell CulturesGmbH] according to the Budapest Convention.

The Rhodoccus strains GF270, GF376, GF473, GF578 and GF674 have beenassigned according to identification to species of the genus Rhodoccuswhich are not yet described in the literature. Accordingly, theinvention also relates to the microorganisms Rhodoccus GF270, RhodoccusGF376, Rhodoccus GF473, Rhodoccus GF578 and Rhodoccus GF674.

Unlike the microorganisms of the genus Actinomadura or Amycolatopsis,the microorganisms of the genus Rhodoccus are expediently induced beforethe actual conversion. Suitable inducers are those described in EP-A-0307 926, such as, for example, acetamide, butyramide, methacrylamide,propionamide, crotonamide and valeramide.

“Functionally equivalent variants and mutants” is understood as meaningmicroorganisms which are derived from the abovementioned sourceorganisms and essentially have the same characteristics and functions asthese. Variants and mutants of this type can be formed by chance, e.g.by UV irradiation or by mutagenic chemicals.

Identification of Amycolatopsis NA40 Colour of aerial mycelium whiteColour of substrate mycelium orange Colour of diffused pigment − Sugarspectrum ARA + GAL + MAD − XYL − GLU tr RIB + Type A DAP DL Menaquinones(in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNA homology 96.9%Phospholipids such as not investigated PE, OH-PE, lyso PE, met PE, PC,NPG, PI, PIM, PG, DPG, GL Fatty acids iso 16 +++ iso 15 + iso 17 (+)anteiso 15 (+) anteiso 17 (+) 10-Me 16 − 10-Me 17 + 2-OH 15 + 2-OH 16 +Type 3f MS − Identification of Amycolatopsis NE31 Colour of aerialmycelium white Colour of substrate mycelium orange Colour of diffusedpigment − Sugar spectrum ARA + GAL + MAD − XYL − GLU tr RIB + Type A DAPDL Menaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNAhomology 96.1% Phospholipids such as not investigated PE, OH-PE, lysoPE, met PE, PC, NPG, PI, PIM, PG DPG, GL Fatty acids iso 16 +++ iso 15 +iso 17 (+) anteiso 15 (+) anteiso 17 (+) 10-Me 16 − 10-Me 17 + 2-OH 15 +2-OH 16 + Type 3f MS − Abbreviations and explanations for theidentification (+) 1–5% + 5–15% ++ 15–30% +++ >30% DAP diaminopimelicacid ARA arabinose GAL galactose MAD madurose XYL xylose GLU glucose RIBribose Sugar types according to Lechevalier et al. 1971 Fatty acid typesaccording to Kroppenstedt 1985 and 1992. 9/4 MK-9 (H₄) 9/6 MK-9 (H₆) 9/8MK-9 (H₈) MS mycolic acids PE phosphatidylethanolamine OH-PE hydroxy-PEmet PE phosphatidimethylethanol- amine PC phosphatidylcholine NPGphosphatidylglucosamine PI phosphatidylinositol PIM phosphatidylinositolmannoside PG phosphatidylglycerol DPG diphosphatidylglycerol GLglycolipids Fatty acids iso-16 isohexadecanoic acids or14-methylpentadecanoic acids 10-Me-18 tuberculostearic acid 2-OH-162-hydroxypalmitic acid

Identification of GF270, GF376, GF473, GF578 and GF674 Theidentification of these strains is based on 5 characteristics which areindependent of one another.

-   -   1. Morphology and colour of the colonies: short-branched hyphae,        which disintegrate into rod- and spore-like elements. The        colonies of GF270 and GF376 are salmon-pink (RAL 3022) and those        of GF578 and GF674 are light red (RAL 3012).    -   2. Diamino acids of the peptidoglycan: meso-diaminopimelic acid    -   3. Mycolic acids: Rhodoccus mycolic acids: The determination of        the long-chain mycolic acid was carried out by means of        high-temperature gas chromatography. The elution profiles of the        mycolic acids of GF270 and GF376 and those of GF473, GF578 and        GF674 were identical. The mycolic acid length for GF270 and        GF376 was C₃₈–C₄₆ and that for GF473, GF578 and GF674 was        C₄₀–C₄₈. The mycolic acid patterns were compared with mycolic        acid patterns of Rhodoccus strains. GF270 was identified with a        very low correlation factor (0.086) as belonging to Rhodococcus        rhodochrous; it was not possible to identify GF376 by this        method. The other three isolates GF473, GF578 and GF674 were        identified with a very low correlation factor as belonging to        Rhodoccus ruber.    -   4. Fatty acid pattern: unbranched, saturated and unsaturated        fatty acids including tuberculostearic acid. The fatty acid        pattern is diagnostic of all Rhodococcus genera and closely        related Mycobacterium, Nocardia, Dietzia, Tsukamurella and some        Corynebacteria species. The identification at the species level        was obtained by qualitative and quantitative differences in the        fatty acid pattern of GF270, GF376, GF473, GF578 and GF674 with        the fatty acid patterns of Rhodoccus species.    -   5. The 16S rDNA subsequences of GF270 and GF376 were identical        (100%), although the comparison of them with the Rhodoccus        strains only showed 99.1% similarity to the closest related        Rhodococcus rhodochrous. GF473 and GF578 were identical in their        16S rDNA sequence (100%). GF674 differs from GF578 in only one        base pair of 500 (99.8%). All three isolates show only a distant        relationship with Rhodoccus coprophilus (98.4%). Based on the        chemotaxic and molecular biology results, it can be concluded        that GF270 and GF376 on the one hand and GF473, GF578 and GF674        on the other hand are strains of 2 new Rhodoccus species. GF270        and GF376 are closely related to Rhodococcus rhodochrous in        their 16S rDNA (99.1%), however GF473, GF578 and GF674 are only        distantly related to Rhodoccus coprophilus (98.4%).

The enzyme extract can be obtained by professionally customarydisruption of the microorganisms, such as, for example, by disruption bymeans of ultrasound, by means of the French press method or the lysozymemethod. This enzyme extract and, of course, also the completemicroorganism cells can be immobilized on a suitable support material,customarily embedded in a polymer, for carrying out the process, orabsorbed on a suitable support material.

The enzymes according to the invention having nitrile hydratase activityare obtainable from the microorganisms of the genus Amycolatopsis andare able to convert a nitrile into an amide, in particular they areobtainable from Amycolatopsis NA40 (DSMZ 11617).

These enzymes in particular have the following properties:

-   a) a pH optimum of pH 6.5±1.0-   b) a temperature optimum between 35 and 40° C. at a pH of 7.0-   c) a K_(M) value for the substrate 3-cyanopyridine of 41.7 mM±7.7 mM    (20° C., 45 mM phosphate buffer, pH 7.0)    in particular the enzymes have a-   d) molecular weight of 106 kDa, such as, for example, determined by    SDS-PAGE.

Nitriles can generally be employed as substrates for thebiotransformation. Expediently, either aliphatic nitriles having 1 to 10carbon atoms, optionally substituted by, for example, hydroxyl, amino,halogen or carboxyl, or substituted or unsubstituted aromatic nitrileshaving 4 to 10 carbon atoms in the aromatic ring system are employed.Aliphatic nitriles having 1 to 10 carbon atoms which can be used aredinitriles, hydroxynitriles, aminonitriles such as, for example,n-octanenitrile, cyanoacetic acid, isocapronitrile, n-valeronitrile,adiponitrile, glutaronitrile, succinonitrile, sebaconitrile,propionitrile, crotononitrile, acrylonitrile, methacrylonitrile,n-butyronitrile or azelanitrile. Aromatic nitriles having 4 to 10 carbonatoms which can be used are nitriles of the general formula

in which R¹ and R² are a hydrogen atom, a halogen atom or C₁₋₄-alkyl. F,Cl, Br or I can be used as halogen atom. Methyl, ethyl, propyl,isopropyl, tert-propyl, butyl, isobutyl or tert-butyl can be used asC₁₋₄-alkyl. Expedient representatives of the aromatic nitriles of thegeneral formula I or II are 2-, 3- or 4-cyanopyridine, benzonitrile,fluoro-, chloro- or bromobenzonitrile, such as, for example, o-, m- orp-chlorobenzonitrile or 2-chloro-3-cyanopyridine. 3-Cyanopyridine ispreferably used as aromatic nitrile having 4 to 10 carbon atoms.

The biotransformation is expediently carried out with addition ofsubstrate in one portion or continuously such that the substrateconcentration does not exceed 40% by weight, preferably 30% by weight.

The process is expediently carried out with resting (non—growing) cells.

Suitable media for the biotransformation are those customary in thespecialist field, such as, for example, low molecular weight phosphatebuffers, HEPES buffers, citrate buffers, borate buffers, the mediaaccording to Tables 1 to 3 or modified forms thereof such as, forexample, those described in Example 8 (1) or TRIS/HCl buffers.

The biotransformation is expediently carried out at a temperature from 0to 50° C. and at a pH between pH 4.5 and pH 10, preferably at atemperature from 20 to 40° C. and at a pH between pH 4.5 and pH 10.0.

In a particularly preferred embodiment, the biotransformation can becarried out in the presence of C₁₋₄-alcohols. C₁₋₄-alcohols employed canbe methanol, ethanol, propanol or butanol. Methanol is preferably used.

After the reaction, the corresponding amides can then be isolated bycustomary working-up methods such as, for example, by crystallization.

EXAMPLES Example 1

Growth of Microorganisms of the Genus Actinomadura or Amycolatopsis

a) Various soil samples were inoculated with various nitriles or amidesas a C and N source in the enrichment medium according to Table 1 andincubated at 37° C. or 45° C. for 7–10 days. The cultures were thentransferred to the same medium and again cultured at 37° C. for 7–10days. The whole process was repeated 3 times. The cultures were thendiluted and plated out in order to obtain individual colonies. Theplates were incubated at 37° C. for 5 days. The different colonies werethen tested for the desired activity.

Amycolatopsis NA40 (DSMZ 11617) and Amycolatopsis NE31 (DSMZ 11616) wereisolated in this way and then grown in the optimized medium (Table 3)for 90–100 h with shaking at 37° C.

Adiponitrile served as a C and N source for Amycolatopsis NE31 (DSMZ11616), Actinomadura spadix E3733 and Actinomadura spadix E3736,azelanitrile served as a C and N source for Amycolatopsis NA40 (DSMZ11617) and Actinomadura spadix 45A32, n-octanenitrile served as a C andN source for Actinomadura spadix 4501 and cyanoacetic acid served as a Cand N source for Actinomadura spadix C15.

b) Amycolatopsis NA40 was cultured in the medium according to Table 3.The culturing was carried out for 2 or 3 to 4 days at a temperature of37° C. under measured turbidimetrically at 610 nm and the dry weight ofthe cells was calculated in the following way: weight of the dry cellsin mg/ml=OD_(610 nm) ×0.277.

TABLE 1 Enrichment medium Nitrile 2.0 g KH₂PO₄ 7.0 g MgSO₄.7H₂O 0.1 gVitamin mixture 1.0 ml CoCl₂.6H₂O 2.0 mg FeSO₄.7H₂O 2.0 mg Make up to 1l with water (pH 6.7–7.3)

TABLE 2 Basal medium Maltose 2.0 g NaNO₃ 1.0 g K₂HPO₄ 0.1 g MgSO₄.7H₂O0.05 g  Make up to 100 ml with water (pH 7.0)

TABLE 3 Optimized medium D-glucose 4.5 g Meat extract 0.5 g K₂HPO₄ 0.1 gMgSO₄.7H₂O 0.05 g CoCl₂.6H₂O 1.0 mg Make up to 100 ml with water (pH7.0)

Example 2

Biotransformations with Microorganisms of the Genus Actinomadura orAmycolatopsis

(1) For determination of the nitrile hydratase activity, a reactionmixture (2 ml) containing 3-cyanopyridine (1.0 M, 1.0 ml), potassiumphosphate buffer (pH 7.0, 0.1 M, 0.5 ml) and 0.5 ml of cell suspensionwere incubated at 20° C. for 30 min with stirring. The reaction wasstopped by addition of 0.2 ml of 3 N HCl. After centrifuging briefly,the nicotinamide formed was determined by means of HPLC (Shimadzu SPD 6Asystem using a C18 column (Develosil ODS-HG-5, 4.6×250 cm); eluent: 10mM KH₂PO₄/H₃PO₄ (pH 2.8)/acetonitrile 9:1 (v/v); flow rate: 1 ml/min;the absorption was measured at 230 nm). The specific activity wasexpressed as μmol of nicotinamide formed/ml/min/OD_(610 nm).

The reaction rates of aliphatic nitriles in the enrichment medium(Table 1) with isolated bacteria is summarized in Table 5, the effectsof inducers and cofactors in the basal medium (Table 2) is summarized inTable 4 and the activity comparison of Amycolatopsis to Rhodoccus in thebasal medium (Table 2) is summarized in Table 6. The results from Table4 show that the nitrile hydratase from Amycolatopsis NA40 isconstitutively expressed but the cofactor cobalt is necessary for theactivity.

(2) Effect of the Temperature on the Growth of NA40

Subcultures (2 ml) were incubated at 37° C. for 2 days in the mediumaccording to Table 3, and then transferred to shaker flasks containing20 ml of medium according to Table 3. Culturing was carried out at 37,40, 45, 50 and 55° C. for 3 to 4 days with shaking. The cell growth wasmeasured and the nitrile hydratase activity was determined at 20° C.Table 7 shows the effect of the temperature on the nitrile hydrataseactivity and on the cell growth.

TABLE 4 Effects of Inducers and cofactors on the specific activity inthe basal medium Total Specific activity activity Growth (μmol/ml/(μmol/ml/ (OD_(610 nm)) min) min/OD) Inducer — 1.26 20.9 16.6 ε-Capro-0.66 9.52 14.5 lactam Crotonamide 3.41 22.9 6.72 Meth- 3.33 2.46 0.74acrylamide Butyramide 2.19 0.19 0.88 Propionamide 1.91 0.92 0.48 Urea1.72 2.97 1.73 Cofactor — 7.97 0.10 0.01 FeSO₄.7H₂O 8.32 3.36 0.40CoCl₂.6H₂O 8.41 47.8 5.68

TABLE 5 Conversion rates of aliphatic nitriles using the isolatedbacteria Growth Total activity Specific activity Strains Substrates(OD_(610 mm)) (μmol/ml/min) (μmol/ml/min/OD) Amycolatopsis NE31 (DSMZAdiponitrile 2.68 0.377 0.141 11616) Actinomadura E3733 Adiponitrile1.62 0.347 0.214 spadix Actinomadura E3736 Adiponitrile 1.36 3.00 2.21spadix Actinomadura 45A32 Azelanitrile 5.81 18.8 3.23 spadixActinomadura 45O1 n-octane- 7.24 32.2 4.45 spadix nitrile ActinomaduraC15 Cyanoacetic 2.04 7.01 3.43 spadix acid Amycolatopsis NA40 (DSMZAzelanitrile 5.92 33.0 5.57 11617)

TABLE 6 Activity of Amycolatopsis in comparison to Rhodococcusrhodochrous J1 Microorganism Amycolatopsis Purified enzyme Purifiedenzyme NA40 Microorganism from NA40 from J1 (DSMZ 11617) Rhodococcus(μmol/min/mg (μmol/min/mg (μmol/ml/min ) rhodochrous J1 protein)protein) Activity for 303 314 1110 371 3-cyanopyridine

(3) For determination of the activity of NA40 with respect to a numberof substrates, cells having a dry weight of 0.0388 mg were incubated inthe buffer described above. The reaction was started by addition of theappropriate substrate and incubated at 20° C. with shaking for 10 min.The reaction was stopped by addition of 0.2 ml of 2 N HCl and thereaction mixture was briefly centrifuged. The supernatant was analysedby means of HPLC or gas chromatography. Table 8 shows the testconditions for the substrate specificity and Table 9 shows the substratespecificity of resting NA40 cells for various substrates.

The respective test conditions are summarized in Table 8 and the resultsare summarized in Table 9.

TABLE 7 Effect of the growth temperature on the nitrile hydrataseactivity and on the cell growth Specific Total activity activity (μmol/Relative Tempera- Growth (μmol/ ml/min/ activity ture (mg/ml) ml/min)mg) (%) 37° C. 6.16 4.69 0.761 100 40° C. 5.79 9.89 1.71 225 45° C. 6.564.83 0.736 97 50° C. 5.96 1.16 0.195 26

TABLE 8 Test conditions for substrate specificity Substrate AnalysisSubstrate (mM) method Amide formed 3-Cyanopyridine 1.0 HPLC Nicotinamide2-Cyanopyridine 0.25 HPLC 2-Picolinamide 4-Cyanopyridine 0.25 HPLCPyridine- 4-carboxamide Crotononitrile 0.4 HPLC Crotonamide Benzonitrile0.03 HPLC Benzamide Acrylonitrile 0.4 HPLC Acrylamide o-Chlorobenzo-0.15 HPLC o-Chlorobenz- nitrile amide m-Chlorobenzo- 0.15 HPLCm-Chlorobenz- nitrile amide p-Chlorobenzo- 0.15 HPLC p-Chlorobenz-nitrile amide 2-Chloro- 0.15 HPLC 2-Chloro- 3-Cyanopyridine nicotinamideAcetonitrile 0.4 GC Acetamide Propionitrile 0.4 GC PropionamideMethacrylo- 0.4 GC Methacrylamide nitrile n-Butyronitrile 0.4 GCn-Butyramide o-, m-, p-Chlorobenzonitrile and 2-chloro-3-cyanopyridinewere added to the reaction mixture dissolved in methanol.

TABLE 9 Substrate specificity of NA40 nitrile hydratase Relativeactivity Substrate (%) 3-Cyanopyridine 100 4-Cyanopyridine 1682-Cyanopyridine 128 Benzonitrile 51 Crotononitrile 52 Acrylonitrile 115o-Chlorobenzo- 96 nitrile m-Chlorobenzo- 75 nitrile p-Chlorobenzo- 16nitrile 2-Chloro- 126 3-cyanopyridine Acetonitrile — Propionitrile 105Methacrylo- 130 nitrile n-Butyronitrile 194

(4) Temperature optimum and thermal stability in resting cells

The reaction was carried out in the standard reaction mixture for 10min. The temperature optimum was between 35 and 40° C. (FIG. 5). Thecells were then incubated at various temperatures for 30 min and theactivity was tested under standard reaction conditions. As can be seenfrom FIG. 4, the heat stability was 40° C.

(5) pH Optimum and pH Stability in Resting Cells

For this purpose, the reaction was carried out for 10 min in thestandard reaction mixture in which the potassium phosphate buffer hadbeen replaced by various 0.1 M buffers. As can be seen from FIG. 6, thepH optimum was between 4.5 and 10. After the cell suspension had beenincubated at 20° C. for 30 min at various pHs, the cells werecentrifuged. The cells were then washed and resuspended in 0.1 Mpotassium phosphate buffer pH 7.0. The reaction was carried out for 10min by addition of 3-cyanopyridine under standard conditions. The enzymewas stable between pH 4.5 and pH 10.0 (FIG. 7).

(6) Accumulation of nicotinamide from 3-cyanopyridine by means of NA40

The reaction was carried out in a reaction mixture (30 ml), comprising500 mM 3-cyanopyridine, 40 mM potassium phosphate buffer (pH 7.0) andresting cells (dry weight 2.3 mg). During the reaction, 3-cyanopyridine(500 mM) was added 7 times as soon as it was consumed. In this manner,4.0 M 3-cyanopyridine was added in the course of 15 h and 3.89 M (475g/l) nicotinamide was formed, corresponding to a yield of 97.3%.Nicotinic acid was not formed.

Example 3

Identification of Microorganisms of the Genus Amycolatopsis

The following 5 chemotaxonomic markers supported the identification:

-   1. Diagnostic amino acid of the peptidoglycan: mesodiaminopimelic    acid-   2. Diagnostic sugars: arabinose and galactose-   3. Mycolic acids: mycolic acids absent-   4. Menaquinones: MK-9 (H₄)-   5. Fatty acid pattern: iso/anteiso-branched and 2-hydroxy fatty    acids, small amounts of 10-methyl-branched fatty acids were    additionally detected. This fatty acid pattern was found in all    representatives of the genus Amycolatopsis (fatty acid pattern 3f)

The combination of these chemical features is diagnostic of all speciesof the genus Amycolatopsis.

The fatty acid data of the two cultures were compared with the aid ofmain component analyses using the entries in the fatty acid database.Using this method, it was possible to assign both NE31 and NA40 to thegenus Amycolatopsis, an identification of the species, however, was notpossible, since the correlation factor was too low. The comparison ofthe fatty acid patterns of both strains showed, however, that they aretwo strains of different types.

The result was confirmed by the results of the 16S rDNA sequenceanalysis.) Here too, assignment to the genus Amycolatopsis took place,but not to any of the Amycolatopsis species described. In this method,the sequence of the 16S rDNA was determined by the direct sequencing ofthe PCR-amplified 16S rDNA gene. The diagnostic part of the 16S rDNAsequence was compared with the sequences of the type species of thegenus Amycolatopsis and related taxa. The result showed that the strainbelongs to the genus Amycolatopsis. The highest agreement was found toAmycolatopsis methanolica at 96.9% (NA40) and 96.1% (NE31). Betweenthem, the two isolates showed agreement in the sequences of 99.0%. Ourinvestigations on representatives of the genus Amycolatopsis have shownthat for a good species identification the correlation factor must behigher than 99.5%. Since at 96.9% the value is clearly below 99.5%, itcan be assumed from this that the two isolates were not representativesof known Amycolatopsis species.

On the basis of the present results, it was not possible to assign theisolates to any of the known Amycolatopsis species. We assume from thisthat NA40 and NE31 are strains of two new, previously undescribedspecies of the genus Amycolatopsis.

Identification Characteristics of Microorganisms of the GenusAmycolatopsis

-   Colour of aerial mycelium-   Colour of substrate mycelium-   Colour of diffused pigment

Sugar spectrum ARA + GAL + MAD − XYL − GLU v RIB + Type A DAP DLMenaquinones (in %) 8/4 − 9/0 (+) 9/2 + 9/4 +++ 9/6 − 9/8 − 16S rDNAhomology >99.5% Phospholipids PE + OH-PE + lyso PE − met PE − PC − NPG −PI + PIM v PG + DPG + GL − Type II + OH-PE Fatty acids iso 16 +++ iso15 + iso 17 (+) anteiso 15 + anteiso 17 (+) 10-Me 16 (+) 10-Me 17 + 2-OH15 + 2-OH 16 + Type 3f MS −

Example 4

Purification of the Nitrile Hydratase from Microorganism Strain NA40

The strain was cultured at 37° C. for 3 days in the medium according toTable 3. The cells of a 2 l culture were harvested by means ofcentrifugation and then resuspended in 0.85% strength NaCl solution. Thecells were then transferred to 0.1 M potassium phosphate buffer (pH 7.0)comprising 44 mM n-butyric acid and treated with ultrasound. The cellextract was centrifuged and the cell fragments were removed. Thisextract was used for the enzyme purification.

During the entire purification, potassium phosphate buffer (pH 7.0)comprising 44 mM n-butyric acid was used. As can be seen from Table 10,the enzyme was purified to homogeneity in 3 steps.

TABLE 10 Purification of the nitrile hydratase from NA40 Total TotalSpecific activity protein activity Enrich- (Units) (mg) (U/mg) mentCell-free 73,300 1020 71.9 1 extract DEAE- 68,000 110 620 8.62 SephacelPhenyl- 64,800 61.4 1105 15.4 TOYOPEARL 1 Unit: The amount of enzymewhich catalyses the formation of 1 μmol of nicotinamide/min at 20° C.

Example 5

Characterization of the Nitrile Hydratase

(1) Determination of the molecular weight, the subunit structure and thecobalt ion content

The molecular weight was determined to be 106 kDa by chromatography on aTSK gel column G3000 SW (0.75×60 cm) using a 0.1 M potassium phosphatebuffer (pH 7.0) containing 0.2 M KC1 and 44 mM n-butyric acid. It wasdetermined that the enzyme consists of 2 different subunits α and β,whose molecular weight was determined to be 30,000 and 26,000 in eachcase.

FIG. 1 shows the determination of the molecular weight by chromatographyon TSK gel G3000 SW.

FIG. 2 shows the determination of the molecular weight by means ofSDS-PAGE

FIG. 3 shows the absorption spectrum of the purified enzyme. A broadabsorption of 300–400 nm was observed.

(2) Substrate Specificity of the Purified Enzyme

The substrate specificity was determined analogously to Example 2 (1).The results are summarized in Table 11.

TABLE 11 Substrate specificity of the purified nitrile hydrataseRelative activity (%) Total Reaction activity with (μmol/ Enzyme restingSubstrate (M) ml/min) reaction cells 3-Cyanopyridine 1.0 17.7 100 1002-Cyanopyridine 0.25 39.1 221 128 4-Cyanopyridine 0.25 31.6 179 168Crotononitrile 0.4 11.9 67 52 Benzonitrile 0.03 11.3 64 51 Acrylonitrile0.4 16.6 94 115 o-Chlorobenzo- 0.15 22.4 127 96 nitrile m-Chlorobenzo-0.15 15.9 90 75 nitrile p-Chlorobenzo- 0.15 2.30 13 16 nitrile 2-Chloro-0.15 16.0 90 126 3-Cyanopyridine Acetonitrile 0.4 — — — Propionitrile0.4 39.3 222 105 Methacrylo- 0.4 22.1 125 130 nitrile n-Butyronitrile0.4 17.9 101 1941.7 Units of enzyme were added to the reaction mixture (2.0 ml). Thereaction mixture contained the respective substrate in 45 mM phosphatebuffer (pH 7.0).(3) Determination of the K_(M) Value

The K_(M) value was determined to be 41.7 mM for 3-cyanopyridine and tobe 3.7 mM for acrylonitrile by means of the Lineweaver-Burk diagram.Compared with Rhodococcus rhodochrous J1, which had a K_(M) valuerelative to 3-cyanopyridine of 200 mM, that of NA40 is significantlylower. This is one of the main advantages of NA40.

(4) Heat Stability and Temperature Optimum

The purified enzyme was incubated for 30 min at pH 7.0 at differenttemperatures and the conversion of 3-cyanopyridine to nicotinamide wasthen measured at 20° C. for 1 min. The enzyme was inactivated at atemperature of greater than 40° C. The heat stability was about 40° C.as in resting cells and the temperature optimum was between 35 and 40°C. (FIG. 5).

(5) pH Optimum and pH Stability

For this purpose, the conversion of 3-cyanopyridine to nicotinamide wascarried out at 20° C. in a reaction mixture (2.0 ml) comprising variousbuffers (42.5 mM), 1.71 units of purified enzyme and 500 mM3-cyanopyridine. The pH optimum was at about pH 6.5±1.0 (FIG. 8).

For determination of the pH stability, 4.2 units of purified enzyme wereincubated at 20° C. for 1 h in various buffers (45 mM). A part of theincubated solution, 1.71 units, were added to the standard reactionmixture (cf. Example 2(1)). The remaining activity was determined. Theenzyme was stable in a pH range from pH 5–9. The result is shown is FIG.9.

(6) Inhibitors

The effect of various inhibitors was determined. The results aresummarized in Table 12.

TABLE 12 Effect of various inhibitors on the purified enzyme RelativeInhibitor mM activity (%) — 100 N-ethylmaleimide 1 97 Iodoacetic acid 139 4-Chloromercurobenzoic acid 0.1 69 Sodium azide 1 59 Hydroxylamine 137 Phenylhydrazine 1 8 Semicarbazide 1 82 Tiron (disodium salt of4,5-dihydroxy-1,3-benzene- 1 110 disulphonic acid o-Phenanthroline 1 89α,α′-Dipyridyl 1 100 8-Hydroxyquinoline 1 110 EDTA(ethylenediaminetetraacetic acid 1 115 Diethyl dithiocarbamate 1 89

Example 6

Effect of Methanol on Resting Cells of NA40

The reaction was carried out for 10 min in the presence of 0–20% (v/v)methanol according to Table 13. As shown in Table 14, the activity isincreased by the addition of 5–15% methanol.

TABLE 13 Reaction with resting cells Methods {circle around (1)} {circlearound (2)} {circle around (3)} {circle around (4)} {circle around (5)}1.0 M 1.0 ml 1.0 ml 1.0 ml 1.0 ml 1.0 ml 3-cyano- pyridine 0.1 M KPB*0.9 ml 0.8 ml 0.7 ml 0.6 ml 0.5 ml (pH 7.0) Methanol — 0.1 ml 0.2 ml 0.3ml 0.4 ml Cell 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml suspension (0.388 mg/ml) Total volume 2.0 ml 2.0 ml 2.0 ml 2.0 ml 2.0 ml *KPB = potassiumphosphate buffer

TABLE 14 Effect of methanol on Amycolatopsis NA40 Methanol Relativeactivity Methods [% (v/v)] [%] {circle around (1)} 0 100 {circle around(2)} 5 123 {circle around (3)} 10 128 {circle around (4)} 15 130 {circlearound (5)} 20 105

Example 7

Enrichment of Microorganisms of the Genus Rhodococcus

Various soil samples were inoculated with cyanoacetic acid as a C and Nsource in the enrichment medium according to Table 1 and themicroorganisms Rhodoccus GF270, GF578, GF473 and GF376 were isolatedaccording to Example 1.

Example 8

Biotransformation using Microorganisms of the Genus Rhodococcus

(1) Heat Stability of the Microorganisms Rhodoccus GF674, RhodoccusGF578, Rhodoccus GF270 and Rhodoccus GF376 in comparison with Rhodoccusrhodochrous J1.

For determination of the heat stability, the microorganisms describedabove were cultured in the following media.

Rhodococcus rhodochrous J1 was cultured for 72 h in the medium describedin EP-A 0 307 926. The microorganisms Rhodoccus GF674, GF578, GF270 andGF376 were cultured in the following media at pH 7.0 for up to 96 h:

Rhodoccus GF674 in a medium comprising yeast extract 1.0 g/l, fructose5.0 g/l, malt extract 10.0 g/l, acetamide 5.0 g/l, KH₂PO₄ 2.0 g/l,MgSO₄. 7H₂O 0.5 g/l and CoCl₂. 6H₂O 10.0 mg. Rhodoccus GF578 in a mediumcontaining yeast extract 1.0 g/l, fructose 15.0 g/l, malt extract 10.0g/l, acetamide 25.0 g/l, KH₂PO₄ 2.0 g/l, MgSO₄. 7H₂O 0.5 g/l and CoCl₂.6H₂O 5.0 mg. Rhodoccus GF270 in a medium containing yeast extract 12.5g/l, sodium citrate 5.0 g/l, methacrylamide 7.5 g/l, KH₂PO₄ 2.0 g/l,MgSO₄. 7H₂O 0.5 g/l and CoCl₂. 6H₂O 30.0 mg. Rhodoccus GF376 in a mediumcontaining yeast extract 1.0 g/l, sodium citrate 10.0 g/l, malt extract15.0 g/l, butyramide 7.5 g/l, KH₂PO₄ 2.0 g/l, MgSO₄. 7H₂O 0.5 g/l andCoCl₂. 6H₂O 15.0 mg.

The resting cells were then incubated for 15 min at various temperaturesand the remaining activity was then determined under the standardreaction conditions according to Example 2(1).

In the course of this it was found that Rhodoccus GF674 had a relativeactivity of nearly 100% at a temperature of 50° C. and stillapproximately an activity of 10% at 60° C. Rhodoccus GF578 likewise had100% relative activity at 50° C. and a relative activity of 20% at 60°C. Rhodoccus GF376 had 100% relative activity up to 50° C., 70% relativeactivity at 60° C. and nearly still 5% relative activity at 70° C.Rhodoccus GF270 had a relative activity of nearly 100% up to 60° C. andlikewise still 5% relative activity at 70° C. In comparison to this,Rhodoccus rhodochrous J1 had 100% relative activity up to 50° C., 80% at60° C. and no longer any activity at 70° C.

In summary, it can therefore be stressed that Rhodoccus GF270 and GF376had a better heat stability than J1 and GF270 had the best heatstability.

(2) pH Optimum of the Rhodococcus Strains

The effect of the pH on the nitrile hydratase activity of the Rhodoccusstrains GF674, GF578, GF270 and GF376 was determined as described inExample 2(5).

The pH optimum of Rhodoccus GF674 was at pH 7.5–9.5, of GF578 at pH8–8.5, for GF270 at pH 6–7.0 and for GF376 at pH 6–8.

(3) Substrate Specificity of the Rhodococcus Strains

The substrate specificity is summarized as relative activity in Table15.

(4) Nicotinamide Accumulation of the Rhodococcus Strains

Analogously to Example 2(6), the Rhodoccus strains GF674, GF578, GF270and GF376 were cultured with 3-cyanopyridine (about 500 mM). In thecourse of this Rhodoccus GF674 formed 6 M nicotinamide within 25 h,GF578 5.5 M nicotinamide within 10 h, GF270 about 8.5 M nicotinamidewithin 20 h and GF376 7.5 M nicotinamide within 20 h.

(5) Tolerance of 3-cyanopyridine on the activity of the Rhodoccusstrains

In order to test the tolerance of 3-cyanopyridine, resting cells wereincubated for 15 min at 20° C. at concentrations of 3-cyanopyridinebetween 1 and 10% (w/v) and the cells were then removed bycentrifugation. After washing the cells with 0.85% NaCl, the remainingactivity was measured.

The tolerance of 3-cyanopyridine as a substrate was tested at varioussubstrate concentrations. It was found that at a substrate concentrationof 2% (w/v) the nitrile hydratase activity of Rhodoccus rhodochrous J1decreased by the factor 1.4, the nitrile hydratase activity of RhodoccusGF674 at a substrate concentration of 4% (w/v) decreased by the factor1.4, the nitrile hydratase activity of Rhodoccus GF578 remained nearlyconstant up to a substrate concentration of 8%, the nitrile hydrataseactivity of Rhodoccus GF270 at a substrate concentration of 4% (w/v)decreased by the factor 1.17 and the nitrile hydratase activity ofRhodoccus GF376 at a substrate concentration of 10% (w/v) decreased bythe factor 1.25.

In comparison with the other Rhodoccus strains, Rhodococcus rhodochrousJi exhibited the poorest tolerance for 3-cyanopyridine.

TABLE 15 Rhodococcus rhodochrous Rhodococcus Rhodococcus RhodococcusRhodococcus J1 GF674 GF578 GF270 GF376 3-Cyanopyridine 100 (%) 100 (%)100 (%) 100 (%) 100 (%) 2-Cyanopyridine 45 308 200 15.7 54.34-Cyanopyridine 70 231 167 55.6 79.8 Benzonitrile 27 138 85.1 13.4 57.22-Chlorobenzonitrile 2.8 64.8 49.0 0 0 3-Chlorobenzonitrile 43 27.8 28.98.41 8.63 4-Chlorobenzonitrile 13 0 0 0 0 Acetonitrile 608 1.49 347 659806 Propionitrile 434 274 132 37.3 245 n-Butyronitrile 352 491 368 181195 Acrylonitrile 478 147 101 192 257 Crotononitrile 78.2 98.0 124 37.1110 Methacrylonitrile 86.9 176 122 0 0

1. An isolated microorganism of the species Rhodoccus GF270 as depositedin the Deutschen Sammlung von Mikroorganismen und Zeilkulturer GmbHandand having accession number DSM
 12211. 2. A microorganism which is amutant of the microorganism of claim 1 which is capable of converting anitrile into an amide.
 3. An isolated microorganism of the speciesRhodoccus GF376 as deposited in the Deutschen Sammlung vonMikroorganismen und Zeilkulturer GmbHand and having accession number DSM12175.
 4. A microorganism which is a mutant of the microorganism ofclaim 3 which is capable of converting a nitrite into an amide.
 5. Aprocess for the preparation of amides, comprising converting a nitrile,as substrate, to the corresponding amide by means of an agent selectedfrom the group consisting of a microorganism of the species RhodoccusGF270 and a mutant thereof.
 6. A process for the preparation of amides,comprising converting a nitrite, as substrate, to the correspondingamide by means of an agent selected from the group consisting of amicroorganism of the species Rhodoccus GF376 and a mutant thereof.
 7. Aprocess according to claim 5 or 6, wherein the nitrite employed is anoptionally substituted aliphatic nitrite having 1 to 10 carbon atoms. 8.A process according to claim 5 or 6, wherein the nitrile employed is anoptionally substituted aromatic nitrile having 4 to 10 carbon atoms inthe aromatic ring system.
 9. A process according to claim 8, wherein thearomatic nitrile is selected from the compounds of the general formula

in which R¹ and R² are a hydrogen atom, a halogen atom or C₁₋₄-alkyl.10. A process according to claim 5 or 6, wherein the reaction is carriedout at a temperature from 0 to 50° C. and at a pH from 4.5 to 10.